G protein-coupled receptors (GPCRs) are involved in a number of physiological signaling processes at both an intracellular and intercellular level. GPCRs are hormone, neurotransmitter and neuromodulator receptors and mediate their intracellular actions through pathways in which G proteins are involved (rev: Kristiansen “Molecular mechanisms of ligand binding, signaling and regulation within the superfamily of G-protein coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function” Pharmacology and Therapeutics, 2004, 103, 21-80). G proteins intracellularly transmit the signal to effector proteins, such as enzymes and ion channels, causing changes in signaling molecules such as cAMP, cGMP, inositol phosphates, diacylglycerol, arachidonic acid and ions. Their activation and regulation is the one of the initial processes of adaptation mechanisms at cell level triggering the activation of second intracellular messengers and the activation of several signaling cascades, phosphorylating enzymes and promoting the regulation at gene level, which will ultimately give rise to a certain physiological effect.
There are two main types of G proteins, heterotrimeric G proteins, binding to GPCRs and participating in intracellular signal transduction mechanisms, and small cytoplasmic G proteins. The former are formed by three subunits, α, β and γ. The βγ subunits are closely associated and can be considered as a single functional unit. With the binding of the agonist, the receptor is activated and undergoes a conformational change resulting in an increase of its affinity for the G protein. This allows a fast GDP dissociation from its binding site in the α subunit. In normal physiological conditions, GDP is immediately replaced by GTP, the concentration of which exceeds that of GDP by several times. The change of guanine nucleotides causes a reduction in the affinity of the α subunit for the βγ complex and the subsequent dissociation of the heterotrimer, in the α subunit on one hand and the βγ dimer on the other hand. Each of the already dissociated subunits can promote the regulation of different second messengers such as 5′-3′ adenosine monophosphate (cAMP) or inositol triphosphate (IP3), and activate different signaling cascades, which results in a great variety of cell functions. The active state lasts until GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the Gα subunits. Once GTP has been hydrolyzed to GDP, the α-GDP and βγ subunits bind again and become inactive.
All the heterotrimeric G proteins follow the same activation/deactivation cycle, thus reversibly allowing a specific intracellular signal transmission. When a GDP molecule binds to the α subunit, the complex is associated with the βγ subunits, thus forming an inactive heterotrimer. Despite the fact that GDP bound to the α subunit can bind to the receptor without βγ, the association with the receptor is highly increased in the presence of βγ.
However, not all the receptors activating G proteins are members of the GPCR superfamily. The activation of G proteins is further involved in the transduction signal mediated by several tyrosine-kinase receptors, such as the epidermal growth factor (EGF) receptor, insulin and growth factors and insulin-like growth factors I and II.
GPCRs are involved in pathologies such as pain, cancer, asthma, inflammation, metabolic, immune, gastrointestinal and neurological disorders. About 500 different GPCRs are known and all of them share the typical molecular structure of 7 hydrophobic domains with about 30 amino acid each traversing the cell membrane, with an extracellular carboxy end and an intracellular amino. The GPCR superfamily comprises receptor for several hormones, neurotransmitters, paracrines and neuromodulators with very important physiological functions. The alteration in the operation of these receptors causes human diseases, and many of these GPCRs are targets for many drugs and abuse drugs. This superfamily includes receptors for several types of endogenous ligands such as amines, peptides, amino acids, glycoproteins, phospholipids, nucleotides, calcium ions, etc. It has been estimated that approximately 80% of known hormones and neurotransmitters activate signal translation mechanisms by means of activating GPCRs, which represent approximately 30-45% of the targets for drugs. GPCRs therefore constitute excellent therapeutic targets for modulating ligand:receptor interactions, which is primarily interesting for developing new drugs.
The development of recombinant DNA techniques has currently allowed obtaining cell preparations overexpressing a certain GPCR subtype. The preparations enriched with membrane receptors are commercially available (for example Amersham Biosciences, PerkinElmer Life and Analytical Sciences), and can be obtained as donations from other research groups or they can be prepared in a cell culture laboratory by means of transfecting cell lines.
GPCRs currently constitute the therapeutic target of more than 30% of the drugs in the market and their sales have produced a large part of the profits of pharmaceutical companies, for example in 2002, the 30 most sold drugs worldwide generated more than 35 billion dollars (Glasel “Emerging Concepts in GPCR research and their implications for drug discovery” Decision Resources 2004).